This invention relates to a process for producing cells of improved competency that are transformable ("competency" referring to the ability of cells to take up and establish exogenous DNA), and to such competent cells. This aspect of the invention relates particularly to cells of Escherichia coli having competency for plasmid DNA. The invention further relates to such a process and to cells produced by the process, which can be repeatedly frozen and thawed without significant loss in transformability.
The methods used for the insertion of exogenous DNA sequences and genes from a variety of organisms into Escherichia coli been described in U.S. Pat. No. 4,237,224 (Cohen and Boyer, 1980). That patent describes the preparation of hybrid plasmid molecules, the insertion of these hybrid plasmid molecules into cells of E. coli, and the replication and amplification of these recombinant plasmids in the transformed cells. The extensive use of and interest in recombinant DNA technology has resulted in a great need for E. coli cells which are capable of taking up and establishing recombinant plasmid molecules, since competent E. coli have become important to the process of introducing and amplifying exogenous genes or sequences.
A number of procedures exist for the preparation of competent E. coli cells and the introduction of DNA into these cells. For example Mandel and Higa (1970, Journal of Molecular Biology 53:159) describe a procedure whereby bacteriophage DNA is combined with E. coli cells in the presence of 50 mM Ca.sup.++ at 0.degree. C. followed by a brief heat pulse at 37.degree.-42.degree. C. This method has been extended to the uptake of chromosomal DNA (Cosloy and Oishi 1973, Proceedings of the National Academy of Science 70:84) and plasmid DNA (Cohen et al 1972, PNAS 69:2110). A summary of the factors influencing the efficiency of transformation is given in Hanahan (1983, JMB 166:557). These factors include the addition of other cations such as Mg, Mn, or Rb to the Ca-treated cells as well as the prolonged incubation of the cells in CaCl.sub.2. The efficiency of transformation of E. coli cells is substantially enhanced by the method described by Hanahan (1983, JMB, hereinafter referred to as "Hanahan (1983)"). The E. coli cells are grown at 37.degree. C. in the presence of 20 mM Mg. Plasmid DNA is combined with the cells at 0.degree. C. in the presence of Mn, Ca, Rb or K, dimethylsulfoxide (DMSO), dithiothreitol (DTT) and hexamine cobalt chloride.
Several E. coli strains prepared by the latter method have transformation efficiencies of from 1 to 5.times.10.sup.8 transformants/ug plasmid DNA. Generally frozen competent cells have transformation efficiencies of about 1.times.10.sup.8 transformants/ug plasmid DNA. These competent cells can be stored frozen at -70.degree. C. for several months without significant loss of transformation efficiency. However, the foregoing frozen cells cannot be thawed and refrozen without significant loss of transformation efficiency.